We hypothesize that increased glutathione reductase activity protects human macrophages from OxLDL-induced mitochondrial dysfunction and cell death, thereby decreasing the severity of atherosclerosis. Macrophage and foam cell death by oncosis plays a crucial role in the development of atherosclerotic lesions. We propose to study the molecular mechanism of glutathione reductase-mediated protection of macrophages from oncosis.Specific Aim 1: To determine the effect of OxLDL on the thiol redox state of mitochondria. Our preliminary data demonstrate that OxLDL induces mitochondrial depolarization and loss of ATP synthesis. We will use human monocyte-derived macrophages to determine if OxLDL promotes oncosis by 1) altering the thiol redox status of mitochondria, 2) inactivating mitochondrial glutathione reductase and 3) increasing mitochondrial inner membrane permeability.Specific Aim 2: To determine the role of mitochondrial and cytosolic glutathione reductase in preventing OxLDL-induced oncosis. Mitochondria do not synthesize glutathione (GSH) and therefore rely on GSH uptake and the reduction of GSSG to maintain the appropriate thiol redox state. We will use human monocyte-derived macrophages to determine 1) if adenovirus-mediated doxycycline-controlled expression of mitochondrial or cytosolic glutathione reductase (GR) prevents GSSG accumulation and protein thiol oxidation and 2) if increasing glutathione reductase activity restores mitochondrial function and protects macrophages from OxLDL-induced oncosis.Specific Aim 3: To determine whether increased macrophage glutathione reductase activity decreases the severity of atherosclerosis. Foam cell death promotes the formation of the necrotic core and the progression of atherosclerotic lesions. We will perform bone marrow transplantation studies to determine in vivo whether augmented expression of glutathione reductase (GR) in macrophages prevents foam cell death and lesion progression. GR-overexpressing bone marrow cells, generated by retroviral gene transfer, will be used to repopulate irradiated LDL receptor null mice and apoE null mice. We will measure both lesion size and lesional cholesterol/cholesterol ester content.